Growth Requirement of

Bacteria, Growth Curve,

Culture Media
Dr. Kohinur Hasan
M.Phil Student(part 1)
Microbiology
Dhaka medical College
What is Bacterial Growth ?
• Growth Refers to the orderly increase in the sum of all the
components of an organism. The increase in size that results when a
cell takes up water or deposits lipid or polysaccharide is not true
growth.
• Microbial Growth Refers to an increase in cell number, not in cell size.
• Bacteria grow and divide by binary fission, a rapid and relatively
simple process by which one parent cell divides to form two progeny
cells.
Total bacterial mass
• 80% Water
• 20% Dry weight=
• Organic matter containing the elements carbon, hydrogen, nitrogen, oxygen,
phosphorus, and sulfur.
• Inorganic ions such as potassium, sodium, iron, magnesium, calcium, and chloride
are required to facilitate enzymatic catalysis and to maintain chemical gradients
across the cell membrane.
Growth requirement of bacteria
• A bacterium requires all the elements in its organic matter as well as
the full complement of ions required for energetics and catalysis.
• In addition, there must be a source of energy to establish the proton
motive force and to allow macromolecular synthesis.
Growth requirement of bacteria
• SOURCES OF METABOLIC ENERGY
• Fermentation
• Respiration
• Photosynthesis
• NUTRITION
• Carbon Source
• Nitrogen Source
• Sulfur Source
• Phosphorus Source
• Mineral Sources
• Growth Factors
Growth requirement of bacteria
• ENVIRONMENTAL FACTORS AFFECTING GROWTH
• Nutrients
• pH
• Temperature
• Aeration
• Ionic Strength and Osmotic Pressure
Fermentation
• Breakdown of suger (glucose, msltose, galactose) to pyruvate and
then lactic acid.

• Also called glycolytic pathway

• Facultative and anaerobic bacteria generate ATP in absence of

oxygen

• Kreb’s cycle produce more ATP than glycolytic pathway.

Respiration
• Respiration is analogous to the coupling of the Energy dependent process to the
discharge of a battery.

• Chemical reduction of an oxidant through a Specific series of electron carrier s in

the membrane establishes the proton motive force across the bacterial membrane.
• reductant (electron donor) may be organic or inorganic (eg, lactic acid serves as a
reductant for some organisms, and hydrogen gas is a reductant for other organisms).
• Gaseous oxygen (O2) is the oxidant most commonly used by aerobic bacteria, but
alternative oxidants that are used by some organisms include carbon dioxide (CO2),
sulfate (SO4 2−), and nitrate (NO3 −).
Photosynthesis
• It is like respiration.

• But the reductant and oxidant are created from photochemically by

light energy absorbed by pigments in the membrane

• Photosynthesis can continue only if there is a source of light energy.

Carbon Source
• Autotroph: Uses photosynthetic energy to reduce carbon dioxide at
the expense of water. They do not require organic nutrients for
growth.
• Chemolithotrophs: organisms that use an inorganic substrate such as
hydrogen or thiosulfate as a reductant and carbon dioxide as a carbon
source.
• Heterotrophs: Require organic carbon for growth, and the organic
carbon must be in a form that can be assimilated.
Nitrogen Source

• Nitrogen is a major component of proteins, nucleic acids, and other

compounds, accounting for approximately 5% of the dry weight of a
typical bacterial cell.
• Source of Nitrogen
Sulfur Source

• It forms part of the structure of several coenzymes and is found in the

cysteinyl and methionyl side chains of proteins.
• Sulfur source: Sulfate and Hydrogen sulfide
Phosphorus Source
• Phosphate (PO43−) is required as a component of ATP, nucleic acids,
and such coenzymes as NAD, NADP, and flavins.
• In addition, many metabolites, lipids (phospholipids, lipid A), cell wall
components (teichoic acid), some capsular polysaccharides, and some
proteins are phosphorylated.
• Phosphate is always assimilated as free inorganic phosphate (Pi).
Mineral Sources
• Magnesium (Mg2+) and ferrous (Fe2+) ions are found in porphyrin
derivatives
• Ca2+ is required as a constituent of Grampositive cell walls
• In formulating a medium for the cultivation of most microorganisms,
it is necessary to provide sources of potassium, magnesium, calcium,
and iron, usually as their ions (K+, Mg2+, Ca2+, and Fe2+). Many other
minerals (eg, Mn2+, Mo2+,Co2+, Cu2+, and Zn2+) are required in
trace quantities—These frequently can be provided in tap water or as
contaminants of other medium ingredients.
Growth Factors
• A growth factor is an organic compound that a cell must have to grow
but that it is unable to synthesize
• Different microbial species vary widely in their growth factor
requirements.
pH
• Most of organisms have narrow optimal pH

• Neutralophiles grow best at a pH of 6.0–8.0

eg. E.coli, Lactobacillus
• Acidophiles have optima as low as pH 3.0
eg. Sulpholobus
• Alkaliphiles have optima as high as pH 10.5
eg.Vibrio cholerae
Temperature
• Temperature
requirements for
growth. Prokaryotes are
commonly divided into
five groups based on
their optimum growth
temperatures. Note that
the optimum
temperature, the point
at which the growth rate
is highest, is near the
upper limit of the range.
Temperature
• Optimal tem is 37 °c for most bacteria

• Psychrophiles: Grow best below 20C. Eg. Pseudomonas

• Mesophiles: Between 25C and 40C. Eg. Most pathogens grow

optimally at 37C

• Thermophiles: Between 50C and 80C. Eg. Geobacillus

Temperature Optima
• Optimum growth
temperature is usually near
the top of the growth range
• Death above the maximum
temp. comes from enzyme
inactivation
• Mesophiles most common
group of organisms
• 40ºF (5°C) slows or stops The relationship of growth rate to temperature for any
given microorganism is seen in a typical Arrhenius plot
growth of most microbes Arrhenius showed that the logarithm of the velocity of
any chemical reaction (log k) is a linear function of
the reciprocal of the temperature (1/T)
Aeration

Oxygen (O2) requirements of prokaryotes

 On the basis of oxygen requirements

• Obligate aerobes: Grow only in presence of oxygen. E.g.

Pseudomonas, Mycobacterium tuberculosis, Bacullus

• Facultative anaerobes: Aerobes, but also grow anaerobically. E.g.

Most pathogens- E. coli, S. aureus.

• Facultative aerobes: Anaerobes, but also grow aerobically. E.g.

Lactobacillus
On the basis of oxygen requirements

• Microaerophillic: grow in 5-10% oxygen, e.g. Campylobacter and

Helicobacter

• Obligate anaerobe: Grow only in absence of oxygen. Eg. Clostridium

• Aerotolerant anaerobe: can tolerate oxygen, but don’t use. Eg.

Clostridium histolyticum
Why anaerobic bacteria are anaerobic?
• are killed by the O 2 ; grow maximally at a pO 2 concentration of less
than 0.5% to 3%.
• a. They lack superoxide dismutase, catalase, and cytochrome-C
oxidase (enzymes that destroy toxic products of oxygen metabolism).
• b. Instead of oxygen, they require another substance such as a
hydrogen acceptor during the generation of metabolic energy and
utilize fermentation pathways with distinctive metabolic products.
Light
• Bacteria except phototrophs grow well in darkness.They are sensitive
to ultraviolet rays and other radiation in light.

• Photochromogenic mycobacteria Produces pigments only in presence

of light.
Ionic Strength & Osmotic Pressure
• A. Hypertonic solutions: High osmotic pressure removes water from
cell, causing shrinkage of cell membrane (plasmolysis). Used to
control spoilage and microbial growth

• B. Hypotonic solutions: Low osmotic pressure causes water to enter

the cell. In most cases cell wall prevents excessive entry of water.
Microbe may lyse or burst if cell wall is weak ( plasmoptysis)
 Ionic Strength & Osmotic Pressure
• Salt concentration

• Halophiles: Require moderate to large salt concentrations. Ocean water

contains 3.5% salt. Eg. Most bacteria in oceans.

• Extreme or Obligate Halophiles: Require very high salt concentrations (20 to

30%). Eg. Bacteria in Dead Sea

• Facultative Halophiles: Do not require high salt concentrations for growth,

but tolerate 2% salt or more.
Microbial Growth
• Bacterial Division: Occurs mainly by binary fission. A few bacterial
species reproduce by budding.
• Generation Time: Time required for a cell to divide, and its population
to double.
• Generation time varies considerably:
• E. coli divides every 20 minutes.
• Most bacteria divide every 1 to 3 hours.
• Some bacteria require over 24 hours to divide.
Logarithmic Representation of Bacterial
Growth

We can express the

number of cells in a
bacterial
generation as 2n,
where n is the number
of doublings that have
occurred.
The Growth Curve
• When bacteria
are inoculated
into a liquid
growth
medium, we
can plot of the
number of
cells in the
population
over time.
Four phases of Bacterial Growth
1. Lag Phase: Period of adjustment to new conditions.

Importance
-Little or no cell division occurs, population size doesn’t increase.
-Phase of intense metabolic activity, in which individual organisms
grow in size.
-May last from one hour to several days.
Four phases of Bacterial Growth

2. Log Phase: Cells begin to divide and generation time reaches a constant minimum.

Importance
-Period of most rapid growth.
-Number of cells produced > Number of cells dying.
-Cells are at highest metabolic activity.
-Cells are most susceptible to adverse environmental factors at this stage - Radiation &
Antibiotics.
-Uniformly stained.
-Produce exotoxin in late stage.
Four phases of Bacterial Growth
3. Stationary Phase: Population size begins to stabilize.

Importance
-Number of cells produced = Number of cells dying.
-Total cell count Slowly increases although the viable count stays constant.
-Cell division begins to slow down. Factors that slow down microbial growth:
Accumulation of toxic waste materials, Acidic pH of media, Limited nutrients
& Insufficient oxygen supply.
-Bacteria become gram variable.
-More exotoxin(1st stage),sporulation, bacteriocins
Four phases of Bacterial Growth

Chemostat:A chemostat is a bioreactor to which fresh medium

is continuously added, while culture liquid containing left over
nutrients, metabolic end products and microorganisms are
continuously removed at the same rate to keep the culture
volume constant.
Four phases of Bacterial Growth
4. Death or Decline Phase: Population size begins to decrease.

Importance
-Number of cells dying > Number of cells produced.
-Cell number decreases at a logarithmic rate.
-Cells lose their ability to divide.
-A few cells may remain alive for a long period of time.
Comparison among four phases
Culture media
• Nutrient material prepared for microbial growth of the laboratory.

• Culture media are required to Isolate the bacteria from the clinical
specimen , following which the appropriate biochemical test ca. Be
performed to identify the causative agent.

• Robert Koch (1843-1910) could be considered the father of culture

media. • His first success in bacteriology was in the isolation of
Bacillus anthracis .
Constituents of Culture Media
• Distilled or potable water with low mineral content
• Electrolytes: NaCl or Others
• Peptone: Complex mixture of partially digested protein
• Source: Protein ( lean mean, heart muscle, soya flour) digested by enzyme like
pepsin
• Constituents: Proteoses, Amino acids, Inorganic Salt, Accessory growth factor
• Special brands: Neopeptone, Proteose peptone, Mycological peptone etc.
Constituents of Culture Media
• Agar: Used for solidifying the culture media. Commercially available in
powder form, melts in water after boiling and jellifies after cooling.
• Components: cell wall derived polysaccharide, fatty acids, inorganic salt
• Preferred over Gelatin
• Agar concentration depends on use
• Meat extract
• Yeast extract
• Malt extract
• Blood and Serum: Important nutrion to fastidious bacteria. Aseptic 5-
10% sheep blood or Sterilized serum
Types of Culture Media
• Based on Consistency
• Liquid media ( or broth)
• Semisolid media
• Solid media
• Based on growth requirements
• Routine laboratory media: prepared from nutrients
• Defined or Synthetic media: Prepared from chemical substance whose
composition is known
Types of Culture Media
• Liquid media :Most commonly used as enrichment where organisms are likely few.
Example:blood culture
advantages:
•  uniform culture conditions
• easy media replacement without changing the container
• sterilization with ultra-filtration and easier container cleaning after use
• Disadvantages:
• -Difficult to isolate different types of bacteria from mixed populations.
• Bacteria grow diffusely in liquids they produce discrete visible growth in solid
media.
• Identification is not possible
Types of Culture Media
• Semisolid media :Prepared by adding small amount of agar to a fluid
medium

Example: transport media (for motility and biochemical test.

Types of Culture Media
• Solid media:  'Agar' is most commonly used to prepare solid
media. Agar is polysaccharide extract obtained from seaweed.
• Example: blood agar, chocolate agar media.

Advantages of solid media:

• (a) Bacteria may be identified by studying the colony character,
• (b) Mixed bacteria can be separated.
• (c)Solid media is used for the isolation of bacteria as pure
culture.
Types of Culture Media
• Disadvantages
• Solid media cannot accommodate large inocula volumes,
typically those greater than 0.05ml.

• they can dehydrate quickly and lose their ability to support

growth within a few days if evaporation prevention measures
are not taken.
Routine laboratory media: based on uses
• Simple/Basal: minimum ingredients for non-fastidious bacteria.
Examples: Peptone water, Nutrients broth/agar,semisolid media

• Nutrient agar media:Mueller-Hinton agar is a microbiological growth

medium that is commonly used for antibiotic susceptibility testing,
Routine laboratory media: based on uses
• Enriched : Basal media + additional nutrient like blood/serum/egg.
Support growth of fastidious and non-fastidious. Examples: Blood
agar, Chocolate agar, Blood culture media
Routine laboratory media: based
Blood Agar:

Composition:

• 5- 10 % sheeps blood to the molten nutrient agar at 45°c

• To test hemolytic property of bacteria (alpha / beta hemolysis)
Eg:streptococci
Routine laboratory media: based on uses
Chocolate agar:
Composition:
Heated blood agar at 70°c
RBCs lysed, colour brown
Eg. Haemophilus influenzae

Loeffler‘s serum slope

Composition: Contain serum

Eg.Corynebacterium diphtheriae

Blood culture media

Brain Heart Infusion broth
Biphasic
Routine laboratory media: based on uses
• Enrichement broth: Liquid media contains inhibitory agents.
Selective and Inhibitory. Helps to separate pathogen from normal
flora in stool ans sputum.

• Examples:
• Tetrathionate broth for Salmonella
• Gram negative broth for isolation of Shigella
• Alkaline peptone water for Vibrio cholarae
Routine laboratory media: based on uses
 Routine laboratory media: based on uses
• Selective media: Solid media containing inhibitory substances that inhibit the normal flora
present in the specimen and allow the pathogens to grow.

• Example:
• Lowenstein Jensen medium for Mycobacterium Tuberculosis

Thiosulfate citrate Bile Salt (TCBS) Mediam for Vibrio

Deoxycholate citrate agar (DCA) and Xylose Lysine Deoxycholate agar  for Salmonella
and Shigella

Potassium tellurite agar (PTA) for C. diphtheriae

Routine laboratory media: based on uses
• Lowenstein Jensen medium
• It is incorporated with congo red and malachite green to inhibit
unwanted bacteria. ... This formulation also encourages the
earliest possible growth of mycobacteria.
• Sterilize by autoclave
Routine laboratory media: based on uses
• Transport media: Bacteria don’t multiply, only remain viable during transport. Example: Pike’s
medium for Streptococcus

• Differential media: Uses indicator to differentiate two groups.

• Example: MacConKey agar differentiate lactose fermenters and non-lactose
fermenters( shigella)
• composition:Peptone
Lactose
Agar
Neutral red
Taurocholate
Sterilize by : autoclave
• Anaerobic culture media: contains reducing substances to permit
growth of obligate anaerobs. Example: Robertson’s Cooked Meat
broth

• Identification media: substrate or chemical are added to help identify

bacteria on primary culture.
• Example:Peptone water sugar, urea broth
Synthetic Media
• Simple synthetic: contains carbon and energy source
(glucose/lactose) and inorganic nitrogen source ( ammonium
chloride) and inorganic salt. Basic essential for non-fastidious
heterotrophs

• Complex synthetic: Simple synthetic plus amino acids/ purines/

pyrimidines and growth factors. Support more exacting bacteria.
Purpose of Culture
• Isolating bacteria from specimen
• To perform biochemical test to identify bacteria
• To perform antimicrobial suspectibility testing
• To maintain stock culture
• For typing of bacterial isolates
• To estimate the viable bacterial count
Methods of cultures
• Loops and Straight wires: are used to inoculate
specimen into culture media.

• Steak Culture: used for isolation of bacteria from

specimen

• Lawn or Carpet Culture: Provides uniformly thick

surface growth of the bacteria on solid medium.
Two methods: Swabbing and Flooding. Used for
carrying out antimicrobial susceptibility testing by
disk diffusion method, bacteriophage typing.
Methods of cultures

• Stroke culture: carried out on agar slopes or

slants by streaking the straight wire in zigzag
fashion. Used for pure growth helpful for
diagnostic test and Urease test

• Stab culture: is done by stabbing the semisolid

agar butt by a straight wire. Used for
maintaining stock culture, demonstration of
oxygen requirements and motility testing
Methods of cultures
• Liquid Culture: Test tube may be inoculated by toching with a loop.
Here growth is detected by observing turbidity in the medium. It
don’t provide pure culture and identification of bacteria not possible.
Methods of cultures

• Pour-plate culture: Quatitative culture method

used to estimate viable bacterial count.
•
• Steps:
• Serial 10-fold dilution
• Pour plating
• Colony Counting
• Viable count/ml
Methods of cultures
• Spread-plate method: Also used to estimate viable bacterial count.
Methods of cultures
• Tryptic Soy Broth (Soybean-Casein Digest Medium) is a general
purpose liquid enrichment medium used in qualitative
procedures for the sterility test and for the enrichment and
cultivation of aerobic microorganisms that are not excessively
fastidious.
Incubation of Culture Media
• Bacteriological incubator device is used for aerobic bacteria.
Inoculated culture plates are incubated at 37C for overnight.
• Candle jar for capnophilic bacteria
• Microaerophilic bacteria require 5% oxygen
Anaerobic culture methods
• Used for Obligate anaerobe.
• Methods:
• Production of Vaccum in a Vaccum desiccator, Inefficient method
• Air evacuation by displacement and combustion of oxygen
• Absorption of oxygen by chemical methods like Alkaline pyrogallol,GasPak
system
• Anaerobic globe box/Chamber
• Anaerobic work station
• Using reducing agents
• Prereduced, anaerobically sterilized (PRAS)
Sterilizing Culture Media
• Autoclaving
Majority of culture media

• Streaming at 100C
Media containing ingredients that would be broken down or inactivated at tem
over 100°c. Eg.Cary-Blair transport medium.

• Filtration
Used mainly to sterilize additives that r heat sensitive and can not be autoclaved or
less stable substancestha to be added to a sterile medium immediately before it used.
Eg- serum and solutions containing urea, carbohydrate.
Preparation of culture media
• Following are imp during preparing culture media

• Environment should be dump free

• Wash the hand immediately after preparing media
• Once tge ingredients are weighted, do not delay in making up the medium
• Use completely clean Glassware, plastic or stainless steel equipment
• Capacity of the container at least twice the volume of tge medium
• Use distilled from a glass still.
• Water containing chloride, lead, copper , detergent must not be used.
• Add powdered or granular ingredients to the water and stir to dessolve, Don’t shake
• Heating carefully
• Autoclaving after ingredients completely dissolved
• Dispensing medium in bottles or tubes
Inoculation
• Sample is placed on sterile medium providing microbes with the
appropriate nutrients to sustain growth.

• Selection of the proper medium and sterility of all tools and media is
important.

• Some microbes may require a live organism or living tissue as the

inoculation medium.
Bacterial inoculation on a culture plate by
inoculation loop
Checking PH of culture medium

• Fluid medium: by dipping narrow pH paper or a pH meter.

• For agar medium: pouring a sample of the molten medium into a

small petri dish .

For dehydrated medium: should not require as it has been prepared

correctly using pure water and clean equipment.
Discard of culture media
• It is suggested that decontamination should be carried out by
autoclaving at 121°C for 60 minutes. After autoclaving, media
may be disposed of through normal waste disposal or
incineration procedures. Media containing selenite should be
disposed of as Special Waste.
References

• Medical Microbiology
• Review of Mefical Microbiology and Immunology
• Essentials of Medical Microbiology by Apurba Sankar Sastry
• Monica District Laboratory
• Thank you